title: STAT3 can be activated through paracrine signaling in breast epithelial cells.
abstract: BACKGROUND
  Many cancers, including breast cancer, have been identified with increased levels of phosphorylated or the active form of Signal Transducers and Activators of Transcription 3 (STAT3) protein. However, whether the tumor microenvironment plays a role in this activation is still poorly understood.
  
  
  METHODS
  Conditioned media, which contains soluble factors from MDA-MB-231 and MDA-MB-468 breast cancer cells and breast cancer associated fibroblasts, was added to MCF-10A breast epithelial and MDA-MB-453 breast cancer cells. The stimulation of phosphorylated STAT3 (p-STAT3) levels by conditioned media was assayed by Western blot in the presence or absence of neutralized IL-6 antibody, or a JAK/STAT3 inhibitor, JSI-124. The stimulation of cell proliferation in MCF-10A cells by conditioned media in the presence or absence of JSI-124 was subjected to MTT analysis. IL-6, IL-10, and VEGF levels were determined by ELISA analysis.
  
  
  RESULTS
  Our results demonstrated that conditioned media from cell lines with constitutively active STAT3 are sufficient to induce p-STAT3 levels in various recipients that do not possess elevated p-STAT3 levels. This signaling occurs through the JAK/STAT3 pathway, leading to STAT3 phosphorylation as early as 30 minutes and is persistent for at least 24 hours. ELISA analysis confirmed a correlation between elevated levels of IL-6 production and p-STAT3. Neutralization of the IL-6 ligand or gp130 was sufficient to block increased levels of p-STAT3 (Y705) in treated cells. Furthermore, soluble factors within the MDA-MB-231 conditioned media were also sufficient to stimulate an increase in IL-6 production from MCF-10A cells.
  
  
  CONCLUSIONS
  These results demonstrate STAT3 phosphorylation in breast epithelial cells can be stimulated by paracrine signaling through soluble factors from both breast cancer cells and breast cancer associated fibroblasts with elevated STAT3 phosphorylation. The induction of STAT3 phosphorylation is through the IL-6/JAK pathway and appears to be associated with cell proliferation. Understanding how IL-6 and other soluble factors may lead to STAT3 activation via the tumor microenvironment will provide new therapeutic regimens for breast carcinomas and other cancers with elevated p-STAT3 levels.
authors.affiliation: Department of Pediatrics, The Ohio State University, Columbus, USA. jlieblein@gmail.com
authors.forename: Jacqueline C
authors.initials: JC
authors.lastname: Lieblein
authors.forename: Sarah
authors.initials: S
authors.lastname: Ball
authors.forename: Brian
authors.initials: B
authors.lastname: Hutzen
authors.forename: A Kate
authors.initials: AK
authors.lastname: Sasser
authors.forename: Huey-Jen
authors.initials: HJ
authors.lastname: Lin
authors.forename: Tim Hm
authors.initials: TH
authors.lastname: Huang
authors.forename: Brett M
authors.initials: BM
authors.lastname: Hall
authors.forename: Jiayuh
authors.initials: J
authors.lastname: Lin
mesh_terms: D001940:Breast / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D050822:Cytokine Receptor gp130 / Q000037:antagonists & inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis; D053612:Janus Kinases / Q000378:metabolism; D010766:Phosphorylation; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D000093242:Interleukin-6 Inhibitors
chemical_list: D017077:Culture Media, Conditioned; D050822:Cytokine Receptor gp130; D015850:Interleukin-6; D053612:Janus Kinases; D050796:STAT3 Transcription Factor; D000093242:Interleukin-6 Inhibitors; C494086:STAT3 protein, human